Low dose methods for treating disorders in which tnf-alpha activity is detrimental

ABSTRACT

A method of treating TNFα disorders is described, wherein the method comprises administering a low dose amount of a TNFα inhibitor.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/421,262, filed Oct. 24, 2002. This application is related to U.S.Pat. Nos. 6,090,382, 6,258,562, and 6,509,015. This application is alsorelated to U.S. patent application Ser. No. 09/801,185, filed Mar. 7,2001; and U.S. patent application Ser. No. 10/302,356, filed Nov. 22,2002. This application is also related to U.S. patent application Ser.No. 10/163,657, filed Jun. 5, 2002, and U.S. patent application Ser. No.10/133,715, filed Apr. 26, 2002. In addition, this application isrelated to U.S. application Ser. No. 10/222,140 and U.S. ProvisionalApplication No. 60/403,907, both of which were filed on Aug. 16, 2002.This application is also related to U.S. Provisional Application Ser.No. 60/397,275, filed Jul. 19, 2002; U.S. Provisional Application Ser.No. 60/411,081, filed Sep. 16, 2002; U.S. Provisional Application Ser.No. 60/417,490, filed Oct. 10, 2002; and U.S. Provisional ApplicationSer. No. 60/455,777, filed Mar. 18, 2003. This application is alsorelated to U.S. patent application Ser. Nos. 10/622,932; 10/623,039;10/623,076; 10/623,065; 10/622,928; 10/623,075; 10/623,035; 10/622,683;10/622,205; 10/622,210; 10/622,683, each of which was filed on Jul. 18,2003. The entire contents of each of these patents and patentapplications are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Tumor necrosis factor α (TNFα) is a cytokine produced by numerous celltypes, including monocytes and macrophages, that was originallyidentified based on its capacity to induce the necrosis of certain mousetumors (see e.g., Old, L. (1985) Science 230:630-632). Subsequently, afactor termed cachectin, associated with cachexia, was shown to be thesame molecule as TNFα. TNFα has been implicated in mediating shock (seee.g., Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57:505-518;Beutler, B. and Cerami, A. (1989) Annu. Rev. Immunol. 7:625-655).Furthermore, TNFα has been implicated in the pathophysiology of avariety of other human diseases and disorders, including sepsis,infections, autoimmune diseases, transplant rejection andgraft-versus-host disease (see e.g., Moeller, A., et al. (1990) Cytokine2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.; European PatentPublication No. 260 610 B1 by Moeller, A., et al. Vasilli, P. (1992)Annu. Rev. Immunol. 10:411-452; Tracey, K. J. and Cerami, A. (1994)Annu. Rev. Med. 45:491-503).

Because of the harmful role of human TNFα (hTNFα) in a variety of humandisorders, therapeutic strategies have been designed to inhibit orcounteract hTNFα activity. In particular, antibodies that bind to, andneutralize, hTNFα have been sought as a means to inhibit hTNFα activity.Some of the earliest of such antibodies were mouse monoclonal antibodies(mAbs), secreted by hybridomas prepared from lymphocytes of miceimmunized with hTNFα (see e.g., Hahn T; et al., (1985) Proc Natl AcadSci USA 82: 3814-3818; Liang, C-M., et al. (1986) Biochem. Biophys. Res.Commun. 137:847-854; Hirai, M., et al. (1987) J. Immunol. Methods96:57-62; Fendly, B. M., et al. (1987) Hybridoma 6:359-370; Moeller, A.,et al. (1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller etal.; European Patent Publication No. 186 833 B1 by Wallach, D.; EuropeanPatent Application Publication No. 218 868 A1 by Old et al.; EuropeanPatent Publication No. 260 610 B1 by Moeller, A., et al.). While thesemouse anti-hTNFα antibodies often displayed high affinity for hTNFα(e.g., Kd≦10⁻⁹M) and were able to neutralize hTNFα activity, their usein vivo may be limited by problems associated with administration ofmouse antibodies to humans, such as short serum half life, an inabilityto trigger certain human effector functions and elicitation of anunwanted immune response against the mouse antibody in a human (the“human anti-mouse antibody” (HAMA) reaction).

In an attempt to overcome the problems associated with use offully-murine antibodies in humans, murine anti-hTNFα antibodies havebeen genetically engineered to be more “human-like.” For example,chimeric antibodies, in which the variable regions of the antibodychains are murine-derived and the constant regions of the antibodychains are human-derived, have been prepared (Knight, D. M, et al.(1993) Mol. Immunol. 30:1443-1453; PCT Publication No. WO 92/16553 byDaddona, P. E., et al.). Additionally, humanized antibodies, in whichthe hypervariable domains of the antibody variable regions aremurine-derived but the remainder of the variable regions and theantibody constant regions are human-derived, have also been prepared(PCT Publication No. WO 92/11383 by Adair, J. R., et al.). However,because these chimeric and humanized antibodies still retain some murinesequences, they still may elicit an unwanted immune reaction, the humananti-chimeric antibody (HACA) reaction, especially when administered forprolonged periods, e.g., for chronic indications, such as rheumatoidarthritis (see e.g., Elliott, M J., et al. (1994) Lancet 344:1125-1127;Elliot, M. J., et al. (1994) Lancet 344:1105-1110).

A preferred hTNFα inhibitory agent to murine mAbs or derivatives thereof(e.g., chimeric or humanized antibodies) would be an entirely humananti-hTNFα antibody, since such an agent should not elicit the HAMAreaction, even if used for prolonged periods. Human monoclonalautoantibodies against hTNFα have been prepared using human hybridomatechniques (Boyle, P., et al. (1993) Cell Immunol. 152:556-568; Boyle,P., at al. (1993) Cell. Immunol. 152:569-581; European PatentApplication Publication No. 614 984 A2 by Boyle, et al.). However, thesehybridoma-derived monoclonal autoantibodies were reported to have anaffinity for hTNFα that was too low to calculate by conventionalmethods, were unable to bind soluble hTNFα and were unable to neutralizehTNFα-induced cytotoxicity (see Boyle, et al.; supra). Moreover, thesuccess of the human hybridoma technique depends upon the naturalpresence in human peripheral blood of lymphocytes producingautoantibodies specific for hTNFα. Certain studies have detected serumautoantibodies against hTNFα in human subjects (Fomsgaard, A., et al.(1989) Scand. J. Immunol. 30:219-223; Bendtzen, K., et al. (1990) Prog.Leukocyte Biol. 10B:447-452), whereas others have not (Leusch, H-G., atal. (1991) J. Immunol. Methods 139:145-147).

Alternative to naturally-occurring human anti-hTNFα antibodies would bea recombinant hTNFα antibody. Recombinant human antibodies that bindhTNFα with relatively low affinity (i.e., K_(d)˜10⁻⁷M) and a fast offrate (i.e., K_(off)˜10⁻² sec⁻¹) have been described (Griffiths, A. D.,at al. (1993) EMBO J. 12:725-734). However, because of their relativelyfast dissociation kinetics, these antibodies may not be suitable fortherapeutic use. Additionally, a recombinant human anti-hTNFα has beendescribed that does not neutralize hTNFα activity, but rather enhancesbinding of hTNFα to the surface of cells and enhances internalization ofhTNFα (Lidbury, A., et al. (1994) Biotechnol. Ther. 5:2745; PCTPublication No. WO 92/03145 by Aston, R. et al.)

Accordingly, human antibodies, such as recombinant human antibodies,that bind soluble hTNFα with high affinity and slow dissociationkinetics and that have the capacity to treat disorders in which TNFαactivity is detrimental, are still needed.

SUMMARY OF THE INVENTION

The invention pertains, at least in part, to methods of administrationof low doses of TNFα inhibitors, including, for example, anti-TNFαantibodies, to treat disorders in which TNFα activity is detrimental.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows arthritic scores of each mouse in the treatment groupsreceiving different doses of D2E7. Arthritic scores were recorded weeklystarting at 1 week of age. For each treatment group, mean±standard errorof arthritis scores are indicated in the graph. The treatment groupswere as follows: Control group: 11 female, 9 male mice (n=20); 10 mg/kgdose group: 2 female, 2 male mice (n=4); 5 mg/kg dose group: 6 female, 1male mice (n=7); 1 mg/kg dose group: 5 female. 3 male mice (n=8); 0.5mg/kg dose group: 3 female, 2 male mice (n=5); 0.1 mg/kg dose group: 3female, 3 male mice (n=6); 0.01 mg/kg dose group: 4 female, 2 male mice(n=6).

FIG. 2 shows arthritic scores of each mouse in the treatment groupsreceiving different doses of Remicade. Arthritic scores were recordedweekly starting at 1 week of age. For each treatment group,mean±standard error of arthritis scores are indicated in the graph. Thetreatment groups were as follows: Control group: 11 female, 9 male mice(n=20); 10 mg/kg dose group: 4 female, 1 male mice (n=5); 5 mg/kg dosegroup: 3 female, 4 male mice (n=7); 1 mg/kg dose group: 6 female, 2 malemice (n=8); 0.5 mg/kg dose group: 4 female, 2 male mice (n=6); 0.1 mg/kgdose group: 1 female, 4 male mice (n=5); 0.01 mg/kg dose group: 2female, 3 male mice (n=5).

FIG. 3 shows arthritic scores of each mouse in the treatment groupsreceiving different doses of Enbrel. Arthritic scores were recordedweekly starting at 1 week of age. For each treatment group,mean±standard error of arthritis scores are indicated in the graph. Thetreatment groups were as follows: Control group: 11 female, 9 male mice(n=20); 10 mg/kg dose group: 3 female, 2 male mice (n=5); 5 mg/kg dosegroup: 3 female, 3 male mice (n=6); 1 mg/kg dose group: 5 female, 1 malemice (n=6); 0.5 mg/kg dose group: 4 female, 3 male mice (n=7); 0.1 mg/kgdose group: 2 female, 3 male mice (n=5); 0.01 mg/kg dose group: 2female, 4 male mice (n=6).

FIG. 4 shows final arthritic scores in D2E7, Remicade, and Enbreltreated huTNF-Tg197 mice at 10 weeks of age.

FIGS. 5A-D show a histopathological evaluation of various tissues takenfrom arthritic joints.

FIGS. 6A, 6B, and 6C show circulating huTNF levels in D2E7, Remicade,and Enbrel treated huTNF-Tg197 mice.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains, at least in part, to low dose methods oftreating disorders in which TNFα activity, e.g., human TNFα activity, isdetrimental. The methods include administering to the subject aneffective amount of a TNFα inhibitor at a low dose, such that thedisorder is treated. The invention also pertains to methods wherein theTNFα inhibitor is administered at a low dose in combination with anothertherapeutic agent and pharmaceutical compositions comprising a TNFαinhibitor, and a pharmaceutically acceptable carrier.

The term “human TNFα” (abbreviated herein as huTNF, hTNFα, or simplyhTNF), as used herein, is intended to refer to a human cytokine thatexists as a 17 kD secreted form and a 26 kD membrane associated form,the biologically active form of which is composed of a trimer ofnoncovalently bound 17 kD molecules. The structure of hTNFα is describedfurther in, for example. Pennica, D., et al. (1984) Nature 312:724-729;Davis, J. M., et al. (1987) Biochemistry 26:1322-1326; and Jones, E. Y.,et al. (1989) Nature 338:225-228. The term human TNFα is intended toinclude recombinant human TNFα (rhTNFα), which can be prepared bystandard recombinant expression methods or purchased commercially (R & DSystems, Catalog No. 210-TA, Minneapolis, Minn.).

The term “TNFα inhibitor” includes agents which inhibit TNFα. Examplesof TNFα inhibitors include etanercept (ENBREL, Immunex), infliximab(REMICADE, Johnson and Johnson), human anti-TNF monoclonal antibody(D2E7, Knoll Pharmaceuticals), CDP 571 (Celltech), and CDP 870(Celltech) and other compounds which inhibit TNFα activity, such thatwhen administered to a subject suffering from or at risk of sufferingfrom a disorder in which TNFα activity is detrimental, the disorder istreated. The term also includes each of the anti-TNFα human antibodiesand antibody portions described herein as well as those described inU.S. Pat. Nos. 6,090,382 and 6,258,562 B1, and in U.S. patentapplication Ser. Nos. 09/540,018, and 09/801,185.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or VL) and a light chain constant region.The light chain constant region is comprised of one domain, CL. The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following orderFR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibodies of the inventionare described in further detail in U.S. Pat. Nos. 6,090,382 and6,258,562 B1, and in U.S. patent application Ser. Nos. 09/540,018, and09/801,185, each of which is incorporated herein by reference in itsentirety.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., hTNFα). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within die term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Theantibody portions of the invention are described in further detail inU.S. Pat. Nos. 6,090,382 and 6,258,562 B1, and in U.S. patentapplication Ser. Nos. 09/540,018, and 09/801,185, each of which isincorporated herein by reference in its entirety.

In one embodiment of the invention, D2E7 antibodies and antibodyportions, D2E7-related antibodies and antibody portions, and other humanantibodies and antibody portions with equivalent properties to D2E7,such as high affinity binding to hTNFα with low dissociation kineticsand high neutralizing capacity are used in low dose methods of treatingdisorders associated with detrimental TNF activity. In another oneembodiment, a human antibody, or an antigen-binding portion thereof,that dissociates from human TNFα with a K_(d) of 1×10⁻⁸ M or less and aK_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determined by surfaceplasmon resonance, and neutralizes human TNFα cytotoxicity in a standardin vitro L929 assay with an IC₅₀ of 1×10⁻⁷ M or less is used in low dosemethods of treating disorders associated with detrimental TNF activity.In a further embodiment, an isolated human antibody, or antigen-bindingportion thereof, dissociates from human TNFα with a K_(off) of 5×10⁻⁴s⁻¹ or less, or even more preferably, with a K_(off) of 1×10⁻⁴ s⁻¹ orless is used in low dose methods of treating disorders associated withdetrimental TNF activity. More preferably, the isolated human antibody,or antigen-binding portion thereof, neutralizes human TNFα cytotoxicityin a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ M or less, evenmore preferably with an IC₅₀ of 1×10⁻⁹ M or less and still morepreferably with an IC₅₀ of 5×10⁻¹⁰ M or less.

The term “low dose” or “low dosage” as used herein, refers to an amountof TNFα inhibitor which is administered to a subject, wherein the amountis substantially lower than that ordinarily employed. A “low dosetherapy” includes a treatment regiment which is based on administering alow dose of a TNFα inhibitor. In one embodiment of the invention, a lowdose of D2E7 is administered to a subject to treat TNFα-associateddisorders which are detrimental. In a further embodiment, a low dose ofTNFα inhibitor, including, for example, D2E7, is used to treatrheumatoid arthritis and symptoms associated with the disease. Forexample, symptoms which can be treated using low dose therapy of D2E7include bone erosion, cartilage erosion, inflammation, and vascularity.Low doses of a TNFα inhibitor are advantageous for a number of reasons,including the reduction in the frequency and severity of side effectswhich may be associated with the normal prescribed dose of TNFαinhibitor.

I. Uses of TNFα Inhibitors of the Invention

In an embodiment, the invention provides a low dose method forinhibiting TNFα activity in a subject suffering from a disorder in whichTNFα activity is detrimental. TNFα has been implicated in thepathophysiology of a wide variety of disorders (see e.g., Moeller, A.,et al. (1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller etal.; European Patent Publication No. 260 610 B1 by Moeller, A). Theinvention provides methods for inhibiting TNFα activity in a subjectsuffering from such a disorder, which method comprises administering tothe subject low dose of an antibody, antibody portion, or other TNFαinhibitor of the invention such that TNFα activity in the subject isinhibited. Preferably, the TNFα is human TNFα and the subject is a humansubject. Alternatively, the subject can be a mammal expressing a TNFαwith which an antibody of the invention cross-reacts. An antibody of theinvention can be administered to a human subject for therapeuticpurposes in low doses (discussed further below). Moreover, a low dose ofan antibody of the invention can be administered to a non-human mammalexpressing a TNFα with which the antibody cross-reacts (e.g., a primate,pig or mouse) for veterinary purposes or as an animal model of humandisease. Regarding the latter, such animal models may be useful forevaluating the therapeutic efficacy of antibodies of the invention(e.g., testing of dosages and time courses of administration).

As used herein, the term “a disorder in which TNFα activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of TNFα in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. For the purposes of the invention, treating adisorder in which TNFα activity is detrimental includes, but is notlimited to, alleviating symptoms associated with said disorder.Accordingly, a disorder in which TNFα activity is detrimental is adisorder in which inhibition of TNFα activity is expected to alleviatethe symptoms and/or progression of the disorder. Such disorders may beevidenced, for example, by an increase in the concentration of TNFα in abiological fluid of a subject suffering from the disorder (e.g., anincrease in the concentration of TNFα in serum, plasma, synovial fluid,etc. of the subject), which can be detected, for example, using ananti-TNFα antibody as described above. There are numerous examples ofdisorders in which TNFα activity is detrimental. The use of a low doseof antibodies, antibody portions, and other TNFα inhibitors of theinvention in the treatment of specific disorders are discussed furtherbelow. In certain embodiments, a low dose of the antibody, antibodyportion, or other TNFα inhibitor of the invention is administered to thesubject in combination with another therapeutic agent, as describedbelow.

A. Sepsis

Tumor necrosis factor has an established role in the pathophysiology ofsepsis, with biological effects that include hypotension, myocardialsuppression, vascular leakage syndrome, organ necrosis, stimulation ofthe release of toxic secondary mediators and activation of the clottingcascade (see e.g., Moeller, A., et al. (1990) Cytokine 2:162-169; U.S.Pat. No. 5,231,024 to Moeller et al.; European Patent Publication No.260 610 B1 by Moeller, A.; Tracey, K. J. and Cerami, A. (1994) Annu.Rev. Med. 4:491-503; Russell, D. and Thompson, R. C. (1993) Curr. Opin.Biotech. 4:714-721). Accordingly, the human antibodies, antibodyportions, and other TNFα inhibitors of the invention can be used totreat sepsis in any of its clinical settings, including septic shock,endotoxic shock, gram negative sepsis and toxic shock syndrome.

Furthermore, to treat sepsis, an anti-hTNFα antibody, antibody portion,or other TNFα inhibitor of the invention can be coadministered with oneor more additional therapeutic agents that may further alleviate sepsis,such as an interleukin-1 inhibitor (such as those described in PCTPublication Nos. WO 92/16221 and WO 92/17583), the cytokineinterleukin-6 (see e.g., PCT Publication No. WO 93/11793) or anantagonist of platelet activating factor (see e.g., European PatentApplication Publication No. EP 374 510). Other combination therapies forthe treatment of sepsis are discussed further in subsection II.

Additionally, in an embodiment, an anti-TNFα antibody, antibody portion,or other TNFα inhibitor of the invention is administered to a humansubject within a subgroup of sepsis patients having a serum or plasmaconcentration of IL-6 above 500 pg/ml, and more preferably 1000 pg/ml,at the time of treatment (see PCT Publication No. WO 95/20978 by Daum,L., et al.).

B. Autoimmune Diseases

Tumor necrosis factor has been implicated in playing a role in thepathophysiology of a variety of autoimmune diseases. For example, TNFαhas been implicated in activating tissue inflammation and causing jointdestruction in rheumatoid arthritis (see e.g., Moeller, A., et al.(1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.;European Patent Publication No. 260 610 B1 by Moeller, A.; Tracey andCerami, supra; Arend, W. P. and Dayer, J-M. (1995) Arth. Rheum.38:151-160; Fava, R. A., et al. (1993) Clin. Exp. Immunol. 94:261-266).TNFα also has been implicated in promoting the death of islet cells andin mediating insulin resistance in diabetes (see e.g., Tracey andCerami, supra; PCT Publication No. WO 94/08609). TNFα also has beenimplicated in mediating cytotoxicity to oligodendrocytes and inductionof inflammatory plaques in multiple sclerosis (see e.g., Tracey andCerami, supra). Chimeric and humanized murine anti-hTNFα antibodies haveundergone clinical testing for treatment of rheumatoid arthritis (seee.g., Elliott, M. J., et al. (1994) Lancet 344:1125-1127; Elliot, M. J.,et al. (1994) Lancet 344:1105-1110; Rankin, E. C., et al. (1995) Br. J.Rheumatol. 34:334-342).

In one embodiment of the invention, low doses of anti-TNFα antibodies ofthe invention can be used to treat rheumatoid arthritis. Low doses ofanti-TNFα antibodies can be used to treat rheumatoid arthritis byalleviating symptoms associated with said disorder. Examples of symptomsor signs commonly associated with rheumatoid arthritis include, but arenot limited to, bone erosion in the joints, cartilage erosion in thejoints, inflammation in the joints, vascularity in the joints, andcombinations thereof. Other examples of symptoms associated withrheumatoid arthritis include weight gain, joint distortion, swelling ofthe joints, joint deformation, ankylosis on felxion, severely impairedmovement, and combinations thereof.

The human antibodies, antibody portions, and other TNFα inhibitors ofthe invention can be used to treat autoimmune diseases, in particularthose associated with inflammation, including rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis and gouty arthritis, allergy,multiple sclerosis, autoimmune diabetes, autoimmune uveitis andnephrotic syndrome. Typically, the antibody, antibody portion, or otherTNFα inhibitor is administered systemically, although for certaindisorders, local administration of the antibody, antibody portion, orother TNFα inhibitor at a site of inflammation may be beneficial (e.g.,local administration in the joints in rheumatoid arthritis or topicalapplication to diabetic ulcers, alone or in combination with acyclohexane-ylidene derivative as described in PCT Publication No. WO93/19751). An antibody, antibody portion, or other TNFα inhibitor of theinvention also can be administered with one or more additionaltherapeutic agents useful in the treatment of autoimmune diseases, asdiscussed further in subsection II.

The antibodies, antibody portions, and other TNFα inhibitors of theinvention, also can be used to treat multisystem autoimmune diseases,including sarcoidosis and Behcet's disease.

C. Infectious Diseases

Tumor necrosis factor has been implicated in mediating biologicaleffects observed in a variety of infectious diseases. For example. TNFαhas been implicated in mediating brain inflammation and capillarythrombosis and infarction in malaria. TNFα also has been implicated inmediating brain inflammation, inducing breakdown of the blood-brainbarrier, triggering septic shock syndrome and activating venousinfarction in meningitis. TNFα also has been implicated in inducingcachexia, stimulating viral proliferation and mediating central nervoussystem injury in acquired immune deficiency syndrome (AIDS).Accordingly, the antibodies, antibody portions, and other TNFαinhibitors of the invention, can be used in the treatment of infectiousdiseases, including bacterial meningitis (see e.g., European PatentApplication Publication No. EP 585 705), cerebral malaria, AIDS andAIDS-related complex (ARC) (see e.g., European Patent ApplicationPublication No. EP 230 574), as well as cytomegalovirus infectionsecondary to transplantation (see e.g., Fietze, E., et al. (1994)Transplantation 58:675-680). The antibodies, antibody portions, or otherTNFα inhibitors of the invention, also can be used to alleviate symptomsassociated with infectious diseases, including fever and myalgias due toinfection (such as influenza) and cachexia secondary to infection (e.g.,secondary to AIDS or ARC).

D. Transplantation

Tumor necrosis factor has been implicated as a key mediator of allograftrejection and graft versus host disease (GVHD) and in mediating anadverse reaction that has been observed when the rat antibody OKT3,directed against the T cell receptor CD3 complex, is used to inhibitrejection of renal transplants (see e.g., Eason, J. D., et al. (1995)Transplantation 52:300-305; Suthanthiran, M. and Strom, T. B. (1994) NewEngl. J. Med. 331:365-375). Accordingly, the antibodies, antibodyportions, and other TNFα inhibitors of the invention, can be used toinhibit transplant rejection, including rejections of allografts andxenografts and to inhibit GVHD. Although the antibody, antibody portion,or other TNFα inhibitor may be used alone, more preferably it is used incombination with one or more other agents that inhibit the immuneresponse against the allograft or inhibit GVHD. For example, in oneembodiment, an antibody, antibody portion, or other TNFα inhibitor ofthe invention is used in combination with OKT3 to inhibit OKT3-inducedreactions. In another embodiment, an antibody, antibody portion, otherTNFα inhibitor of the invention is used in combination with one or moreantibodies directed at other targets involved in regulating immuneresponses, such as the cell surface molecules CD25 (interleukin-2receptor-α), CD11a (LFA-1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80(B7-1) and/or CD86 (B7-2). In yet another embodiment, an antibody,antibody portion, or other TNFα inhibitor of the invention is used incombination with one or more general immunosuppressive agents, such ascyclosporin A or FK506.

E. Malignancy

Tumor necrosis factor has been implicated in inducing cachexia,stimulating tumor growth, enhancing metastatic potential and mediatingcytotoxicity in malignancies. Accordingly, the antibodies, and antibodyportions, of the invention, can be used in the treatment ofmalignancies, to inhibit tumor growth or metastasis and/or to alleviatecachexia secondary to malignancy. The antibody, antibody portion, otherTNFα inhibitor may be administered systemically or locally to the tumorsite.

The antibodies, antibody portions, and other TNFα inhibitors of theinvention, also can be used to treat malignant disorders associated withsolid tumors and/or leukemias and lymphomas. Examples of solid tumorswhich can be treated with the antibodies of the invention include, butare not limited to, ovarian cancer and colorectal cancer. Examples ofleukemias and lymphomas which can be treated with the antibodies of theinvention include, but are not limited to, myelo dysplastic syndrome andmultiple myeloma.

F. Pulmonary Disorders

Tumor necrosis factor has been implicated in the pathophysiology ofadult respiratory distress syndrome (ARDS), including stimulatingleukocyte-endothelial activation, directing cytotoxicity to pneumocytesand inducing vascular leakage syndrome. Accordingly, the antibodies,antibody portions, and other TNFα inhibitors of the invention, can beused to treat various pulmonary disorders, including adult respiratorydistress syndrome (see e.g., PCT Publication No. WO 91/04054), shocklung, chronic pulmonary inflammatory disease, pulmonary sarcoidosis,pulmonary fibrosis, asilicosis, asthma, chronic obstructive pulmonarydisease (COPD), and idiopathic pulmonary fibrosis (UIP or interstitiallung disease). The antibody, antibody portion, or other TNFα inhibitormay be administered systemically or locally to the lung surface, forexample as an aerosol. An antibody, antibody portion, or other TNFαinhibitor of the invention also can be administered with one or moreadditional therapeutic agents useful in the treatment of pulmonarydisorders, as discussed further in subsection II.

G. Intestinal Disorders

Tumor necrosis factor has been implicated in the pathophysiology ofinflammatory bowel disorders (see e.g., Tracy, K. J., et al. (1986)Science 234:470-474; Sun, X-M., et al. (1988) J. Clin. Invest.L81:1328-1331; MacDonald, T. T., et al. (1990) Clin. Exp. Immunol.81:301-305). Chimeric murine anti-hTNFα antibodies have undergoneclinical testing for treatment of Crohn's disease (van Dullemen, H. M.,et al. (1995) Gastroenterology 109:129-135). The human antibodies,antibody portions, and other TNFα inhibitors of the invention, also canbe used to treat intestinal disorders, such as idiopathic inflammatorybowel disease, which includes two syndromes, Crohn's disease andulcerative colitis. An antibody, antibody portion, and other TNFαinhibitors of the invention also can be administered with one or moreadditional therapeutic agents useful in the treatment of intestinaldisorders, as discussed further in subsection H.

H. Cardiac Disorders

The antibodies, antibody portions, and other TNFα inhibitors of theinvention, also can be used to treat various cardiac disorders,including ischemia of the heart (see e.g., European Patent ApplicationPublication No. EP 453 898) and heart insufficiency (weakness of theheart muscle) (see e.g., PCT Publication No. WO 94/20139).

The antibodies, antibody portions, and other TNFα inhibitors of theinvention, also can be used to treat cardiovascular disorders including,but not limited to, chronic artherosclerosis, cardiomyopathy, congestiveheart failure, and rheumatic heart disease.

I. Neurological Disorders The antibodies, antibody portions, and otherTNFα inhibitors of the invention, can be used to treat neurologicaldisorders, including, for example, Alzheimer's, Sciatica, peripheralneuropathy, and neuropathic pain.

J. Metabolic Disease

Tumor necrosis factor has been implicated in mediating biologicaleffects observed in a variety of metabolic diseases. For example, theantibodies, antibody portions, and other TNFα inhibitors of theinvention can be used to treat cachexia.

Tumor necrosis factor has also been implicated in mediating thebiological effects observed in diabetes and complications associatedwith diabetes. Diabetic conditions include, but are not limited to, type1 diabetes mellitus, type 2 diabetes mellitus, diabetic vasculopathy,and neuropathic pain.

K. Liver Disease

Tumor necrosis factor has been implicated in mediating biologicaleffects observed in a variety of liver diseases. The antibodies,antibody portions, and other TNFα inhibitors of the invention, can beused to treat liver diseases, including, for example, hepatitis C,schlerosing cholangitis, autoimmune hepatitis, and chronic liverfailure.

L. Kidney Disease

Tumor necrosis factor has been implicated in mediating biologicaleffects observed in a variety of kidney diseases. The antibodies,antibody portions, and other TNF inhibitors of the invention, can beused to treat kidney diseases, including, for example progressive renalfailure. The antibodies of the invention can also be used to treatglomerulonephrities, including, for example, post-streptococcalglomerulonephritis and IgA nephropathy.

M. Inflammatory Disease

1. Inflammatory Joint Disease

The antibodies, antibody portions, and other TNFα inhibitors of theinvention, also can be used to treat inflammatory joint disease,including, for example, Adult Still's disease, juvenile rheumatoidarthritis, Still's disease, Reiter's syndrome, andspondyloarthropathies. The antibodies of the invention can also be usedto treat spondyloarthropathies. Examples of spondyloarthropathiesinclude, for example, ankylosing spondylitis, psoriatic arthritis, andundifferentiated spondyloarthropathies.

2. Inflammatory Connective Tissue Disease

The antibodies, antibody portions, and other TNFα inhibitors of theinvention, also can be used to treat inflammatory connective tissuediseases, including, for example, dermato/polymyositis, scleroderma,mixed connective tissue disorder, relapsing polychondritis, andvascultides. Examples of vascultides include Wegener's granulomatosis,temporal arteritis (GCA) and polymyalgia rheumatica. Takayasu'sarteritis, polyarteritis nodosa, microscopic polyangiitis, Churg-Strausssyndrome, and Kawasaki syndrome.

3. Inflammatory Skin and Mucosal Diseases

The antibodies, antibody portions, and other TNFα inhibitors of theinvention, also can be used to treat inflammatory skin and mucosaldiseases, including, for example, psoriasis, pemphigus vulgaris,Jarisch-Herxheimer reaction, pyoderma gangerenosum, and drug reactionssuch as erythema multiforme and Stevens Johnson syndrome.

4. Inflammatory Diseases of Sensory Organs

The antibodies, antibody portions, and other TNFα inhibitors of theinvention, can be used to treat inflammatory diseases of the sensoryorgans, including uveitis and autoimmune hearing loss. The antibodies ofthe invention can also be used to treat inflammatory diseases associatedwith the ear, including chronic otitis media with or withoutcholesteatoma, chronic ear inflammation, and pediatric ear inflammation.Clinical studies have shown that cytokines, including TNFα, areupregulated in patients with chronic otitis media with cholesteatoma(Yetiser et al. (2002) Otology and Neurotology 23: 647-652). Theantibodies of the invention can be used to treat inflammation andcholesteatoma associated with otitis media.

5. Inflammatory/Autoimmunne Diseases of Other Organ Systems

The antibodies, antibody portions, and other TNFα inhibitors of theinvention, can be used to treat inflammatory/autoimmune diseases ofother organ systems, including, for example, familial periodic fevers,prostatitis, Felty's syndrome, Sjogren's syndrome, acute pancreatitis,chronic pancreatitis, and orchitis.

N. Degenerative Bone and Joint Disease

The antibodies, antibody portions, and other TNFα inhibitors of theinvention, can be used to treat various disorders associated withdegenerative bone and joint disease, including, for example, pseudogout,ostoarthritis, periodontal disease, and loosening of prostheses, e.g.artificial hips (metallic head of femur, etc.) or osteolysis.

O. Reperfusion Injury

The antibodies, antibody portions, and other TNFα inhibitors of theinvention, can be used to treat various disorders associated withreperfusion injury, including, for example, stroke and myocardialinfarction.

P. Others

The antibodies, and antibody portions, of the invention, also can beused to treat various other disorders in which TNFα activity isdetrimental. Examples of other diseases and disorders in which TNFαactivity has been implicated in the pathophysiology, and thus which canbe treated using an antibody, antibody portion, or other TNFα inhibitorof the invention, include inflammatory bone disorders and boneresorption disease (see e.g., Bertolini, D. R., et al. (1986) Nature319:516-518; Konig, A., et al. (1988) J. Bone Miner. Res. 3:621-627;Lerner, U. H. and Ohlin, A. (1993) J. Bone Miner. Res. 8:147-155; andShankar, G. and Stern, P. H. (1993) Bone 14:871-876), hepatitis,including alcoholic hepatitis (see e.g., McClain, CJ. and Cohen, D. A.(1989) Hepatology 9:349-351; Felver, M. E., et al. (1990) Alcohol. Clin.Exp. Res. 14:255-259; and Hansen, J., et al. (1994) Hepatology20:461-474), viral hepatitis (Sheron, N., et al. (1991) J. Hepatol.12:241-245; and Hussain, M. J., et al. (1994) J. Clin. Pathol.47:1112-1115), and fulminant hepatitis; coagulation disturbances (seee.g., van der Poll, T., et al. (1990) N. Engl. J. Med. 322:1622-1627;and van der Poll, T., et al. (1991) Prog. Clin. Biol. Res. 367:55-60),burns (see e.g., Giroir, B. P., et al. (1994) Am. J. Physiol.267:H118-124; and Liu, X. S., et al. (1994) Burns 20:40-44), reperfusioninjury (see e.g., Scales, W. E., et al. (1994) Am. J. Physiol.267:G1122-1127; Serrick, C., et al. (1994) Transplantation 58:1158-1162;and Yao, Y. M., et al. (1995) Resuscitation 29:157-168), keloidformation (see e.g., McCauley, R. L., et al. (1992) J. Clin. Immunol.12:300-308), scar tissue formation; pyrexia; periodontal disease;obesity and radiation toxicity.

Other disorders in which TNFα activity is detrimental include, but arenot limited to, hepatotoxicity, adult Still's disease, Alzheimer'sdisease, ankylosing spondylitis, asthma, cancer and cachexia,atherosclerosis, chronic atherosclerosis, chronic fatigue syndrome,liver failure, chronic liver failure, obstructive pulmonary disease,chronic obstructive pulmonary disease, congestive heart failure,dermatopolymyositis, diabetic macrovasculopathy, endometriosis, familialperiodic fevers, fibrosis, hemodialysis, Jarisch-Herxheimer reaction,juvenile RA, Kawasaki syndrome, myelo dysplastic syndrome, myocardialinfarction, panciaticular vulgaris, periodontal disease, peripheralneuropathy, polyarticular, polymyositis, progressive renal failure,psoriasis, psoriatic arthritis, Reiter's syndrome, sarcoidosis,scleroderma, spondyloarthropathies, Still's disease, stroke, therapyassociated syndrome, therapy induced inflammatory syndrome, inflammatorysyndrome following IL-2 administration, thoracoabdominal aortic aneurysmrepair (TAAA), Vasulo-Behcet's disease, Yellow Fever vaccination, type 1diabetes mellitus, type 2 diabetes mellitus, neuropathic pain, sciatica,cerebral edema, edema in and/or around the spinal cord, vasculitide,Wegener's granulomatosis, temporal arteritis, polymyalgia rheumatica,Takayasu's arteritis, polyarteritis nodosa, microscopic polyangiitis,Churg-Strauss syndrome, Felty's syndrome, Sjogren's syndrome, mixedconnective tissue disorder, relapsing polychondritis, pseudogout,loosening of prostheses, autoimmune hepatitis, sclerosing cholangitis,acute pancreatitis, chronic pancreatitis, glomerulonephritides,post-streptococcal glomerulonephritis or IgA nephropathy, rheumaticheart disease, cardiomyopathy, orchitis, pyoderma gangerenosum, multiplemyeloma, TNF receptor associated periodic syndrome [TRAPS],atherosclerosis, steroid dependent giant cell arteritismyostitis,uveitis, and drug reactions.

II. Pharmaceutical Compositions and Pharmaceutical Administration

The antibodies, antibody-portions, and other TNFα inhibitors of theinvention can be incorporated into pharmaceutical compositions suitablefor low dose administration to a subject. Typically, the pharmaceuticalcomposition comprises an antibody, antibody portion, or other TNFαinhibitor of the invention and a pharmaceutically acceptable carrier. Asused herein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the antibody,antibody portion, or other TNFα inhibitor.

The compositions of this invention may be in a variety of forms suitablefor low dose administration. These include, for example, liquid,semi-solid and solid dosage forms, such as liquid solutions (e.g.,injectable and infusible solutions), dispersions or suspensions,tablets, pills, powders, liposomes and suppositories. The preferred formdepends on the intended mode of administration and therapeuticapplication. Typical preferred compositions are in the form ofinjectable or infusible solutions, such as compositions similar to thoseused for passive immunization of humans with other antibodies or otherTNFα inhibitors. The preferred mode of administration is parenteral(e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In apreferred embodiment, a low dose of the antibody or other TNFα inhibitoris administered by intravenous infusion or injection. In anotherpreferred embodiment, a low dose of the antibody or other TNFα inhibitoris administered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating a low dose of the activecompound (i.e., antibody, antibody portion, or other TNFα inhibitor) inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

The invention also pertains to packaged pharmaceutical compositionswhich comprise a low dose of a TNFα inhibitor of the invention andinstructions for using the inhibitor to treat a particular disorder inwhich TNFα activity is detrimental, as described above.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” is any amount which is determined tobe required to eliminate said disorder or to reduce and/or alleviate thesymptoms of said disorder. In a preferred embodiment of the invention, a“therapeutically effective amount” refers to an amount which iseffective, at low doses and for periods of time necessary, to achievethe desired therapeutic result. A therapeutically effective amount ofthe antibody, antibody portion, or other TNFα inhibitor may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the antibody, antibody portion, otherTNFα inhibitor to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody, antibody portion, or other TNFαinhibitor are outweighed by the therapeutically beneficial effects.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided low doses may be administeredover time or the low dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in lowdosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic or prophylactic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody, antibody portion, orother TNFα inhibitor of the invention is 0.01-2.0 mg/kg. It is to benoted that dosage values may vary with the type and severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

In one embodiment of the invention, the therapeutically effective amountof an anti-TNFα antibody is a low dose. In one embodiment, the low doseof the antibody administered to a subject suffering from a disorder inwhich TNFα is detrimental, is between about 0.01-2.0 mg/kg, about0.06-1.9 mg/kg, about 0.11-1.8 mg/kg, about 0.16-1.7 mg/kg, about0.21-1.6 mg/kg, about 0.26-1.5 mg/kg, about 0.31-1.4 mg/kg, about0.36-1.3 mg/kg, about 0.41-1.2 mg/kg, about 0.46-1.1 mg/kg, about0.51-1.0 mg/kg, about 0.56-0.9 mg/kg, about 0.61-0.8 mg/kg, and about0.66-0.7 mg/kg. In a preferred embodiment, the antibody is D2E7. Rangesintermediate to the above recited dosages, e.g. about 0.17-1.65 mg/kgare also intended to be part of this invention. For example, ranges ofvalues using a combination of any of the above recited values as upperand/or lower limits are intended to be included.

A low dose of the antibodies, antibody-portions, and other TNFαinhibitors of the present invention can be administered by a variety ofmethods known in the art, although for many therapeutic applications,the preferred route/mode of administration is intravenous injection orinfusion. As will be appreciated by the skilled artisan, the routeand/or mode of administration will vary depending upon the desiredresults. In certain embodiments, the active compound may be preparedwith a carrier that will protect the compound against rapid release,such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, a low dose of an antibody, antibody portion; orother TNFα inhibitor of the invention may be orally administered, forexample, with an inert diluent or an assimilable edible carrier. Thecompound (and other ingredients, if desired) may also be enclosed in ahard or soft shell gelatin capsule, compressed into tablets, orincorporated directly into the subject's diet. For oral therapeuticadministration, the compounds may be incorporated with excipients andused in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Toadminister a low dose of the compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, a low dose of an antibody orantibody portion of the invention is coformulated with and/orcoadministered with one or more additional therapeutic agents that areuseful for treating disorders in which TNFα activity is detrimental. Forexample, a low dose of an anti-hTNFα antibody, antibody portion, orother TNFα inhibitor of the invention may be coformulated and/orcoadministered with one or more additional antibodies that bind othertargets (e.g., antibodies that bind other cytokines or that bind cellsurface molecules), one or more cytokines, soluble TNFα receptor (seee.g., PCT Publication No. WO 94/06476) and/or one or more chemicalagents that inhibit hTNFα production or activity (such ascyclohexane-ylidene derivatives as described in PCT Publication No. WO93/19751). Furthermore, a low dose of one or more antibodies or otherTNFα inhibitors of the invention may be used in combination with two ormore of the foregoing therapeutic agents. Such combination therapies mayadvantageously utilize even lower dosages of the administeredtherapeutic agents, thus avoiding possible toxicities or complicationsassociated with the various monotherapies.

III. Other Therapeutic Agents

The language “in combination with” a therapeutic agent includesco-administration of a low dose of the antibody, antibody portion, orother TNFα inhibitor of the invention with a therapeutic agent,administration of a low dose of the antibody, antibody portion, or otherTNFα inhibitor of the invention first, followed by the therapeutic agentand administration of the therapeutic agent first, followed by the lowdose of the antibody, antibody portion, or other TNFα inhibitor of theinvention. Specific therapeutic agent(s) are generally selected based onthe particular disorder being treated, as discussed below.

Nonlimiting examples of therapeutic agents for rheumatoid arthritis withwhich a low dose of an antibody, antibody portion, or other TNFαinhibitor of the invention can be combined include the following:non-steroidal anti-inflammatory drug(s) (NSAIDs); cytokine suppressiveanti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanizedanti-TNFα antibody; Celltech/Bayer); cA2 (chimeric anti-TNFα antibody;Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein;Immunex; see e.g., Arthritis & Rheumatism (1994) Vol. 37, S295; J.Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNFreceptor-IgG fusion protein; Hoffmann-LaRoche); IDEC-CE9.1/SB 210396(non-depleting primatized anti-CD4 antibody; IDEC/SmithKline; see e.g.,Arthritis & Rheumatism (1995) Vol. 31, S185); DAB 486-IL-2 and/or DAB389-IL-2 (IL-2 fusion proteins; Seragen; see e.g., Arthritis &Rheumatism (1993) Vol. 36, 1223); Anti-Tac (humanized anti-IL-2Rα;Protein Design Labs/Roche); IL-4 (anti-inflammatory cytokine;DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10, anti-inflammatorycytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4 agonists (e.g.,agonist antibodies); IL-IRA (IL-1 receptor antagonist; Synergen/Amgen);TNF-bp/s-TNFR (soluble TNF binding protein; see e.g., Arthritis &Rheumatism (1996) Vol. 39. No. 9 (supplement), S284; Amer. J.Physiol.—Heart and Circulatory Physiology (1995) Vol. 268, pp. 37-42);R973401 (phosphodiesterase Type IV inhibitor; see e.g., Arthritis &Rheumatism (1996) Vol. 29, No. 9 (supplement), S282); MK-966 (COX-2Inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S81); Iloprost (see e.g., Arthritis & Rheumatism (1996)Vol. 39, No. 9 (supplement), S82); methotrexate; thalidomide (see e.g.,Arthritis & Rheumatism (1996) Vol. 2, No. 9 (supplement), S282) andthalidomide-related drugs (e.g., Celgen); leflunomide (anti-inflammatoryand cytokine inhibitor, see e.g., Arthritis & Rheumatism (1996) Vol. 39,No. 9 (supplement), S131; Inflammation Research (1996) Vol. 45, pp.103-107); tranexamic acid (inhibitor of plasminogen activation; seee.g., Arthritis & Rheumatism (1996) Vol. 22, No. 9 (supplement), S284);T-614 (cytokine inhibitor, see e.g., Arthritis & Rheumatism (1996) Vol.29, No. 9 (supplement), S282); prostaglandin E1 (see e.g., Arthritis &Rheumatism (1996) Vol. 22, No. 9 (supplement), S282); Tenidap(non-steroidal anti-inflammatory drug; see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S280); Naproxen (non-steroidalanti-inflammatory drug; see e.g., Neuro Report (1996) Vol. 7, pp.1209-1213); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen(non-steroidal anti-inflammatory drug); Piroxicam (non-steroidalanti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatorydrug); Indomethacin (non-steroidal anti-inflammatory drug);Sulfasalazine (see e.g., Arthritis & Rheumatism (1996) Vol. 29, No. 9(supplement), S281); Azathioprine (see e.g., Arthritis & Rheumatism(1996) Vol. 32, No. 9 (supplement), S281); ICE inhibitor (inhibitor ofthe enzyme interleukin-1β converting enzyme); zap-70 and/or lckinhibitor (inhibitor of the tyrosine kinase zap-70 or lck); VEGFinhibitor and/or VEGF-R inhibitor (inhibitos of vascular endothelialcell growth factor or vascular endothelial cell growth factor receptor;inhibitors of angiogenesis); corticosteroid anti-inflammatory drugs(e.g., SB203580); TNF-convertase inhibitors; anti-IL-12 antibodies;interleukin-11 (see e.g., Arthritis & Rheumatism (1996) Vol. 32, No. 9(supplement). S296); interleukin-13 (see e.g., Arthritis & Rheumatism(1996) Vol. 3, No. 9 (supplement), S308); interleukin-17 inhibitors (seee.g., Arthritis & Rheumatism (1996) Vol. 29, No. 9 (supplement), S120);gold; penicillamine; chloroquine; hydroxychloroquine; chlorambucil;cyclophosphamide; cyclosporine: total lymphoid irradiation;anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins;orally-administered peptides and collagen; lobenzarit disodium; CytokineRegulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals.Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10;T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycanpolysulphate; minocycline; anti-IL2R antibodies; marine and botanicallipids (fish and plant seed fatty acids; see e.g., DeLuca et al. (1995)Rheum. Dis. Clin. North Am. 21:759-777); auranofin; phenylbutazone;meclofenamic acid; flufenamic acid; intravenous immune globulin;zileuton; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus(rapamycin); amiprilose (therafectin); cladribine(2-chlorodeoxyadenosine); and azaribine.

Nonlimiting examples of therapeutic agents for inflammatory boweldisease with which a low dose of an antibody, antibody portion, otherTNFα inhibitor of the invention can be combined include the following:budenoside; epidermal growth factor; corticosteroids; cyclosporin,sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine;metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine;balsalazide; antioxidants: thromboxane inhibitors; IL-1 receptorantagonists; anti-IL-1β monoclonal antibodies; anti-IL-6 monoclonalantibodies; growth factors; elastase inhibitors; pyridinyl-imidazolecompounds; CDP-571/BAY-10-3356 (humanized anti-TNFα antibody;Celltech/Bayer); cA2 (chimeric anti-TNFα antibody; Centocor); 75kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see e.g.,Arthritis & Rheumatism (1994) Vol. 3, S295; J. Invest. Med. (1996) Vol.44, 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein;Hoffmann-LaRoche); interleukin-10 (SCH 52000; Schering Plough); IL-4;IL-10 and/or IL-4 agonists (e.g., agonist antibodies); interleukin-11;glucuronide- or dextran-conjugated prodrugs of prednisolone,dexamethasone or budesonide; ICAM-1 antisense phosphorothioateoligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); solublecomplement receptor 1 (TP10; T Cell Sciences, Inc.); slow-releasemesalazine; methotrexate; antagonists of Platelet Activating Factor(PAF); ciprofloxacin; and lignocaine.

Nonlimiting examples of therapeutic agents for multiple sclerosis withwhich a low dose of an antibody, antibody portion, or other TNFαinhibitors of the invention can be combined include the following:corticosteroids; prednisolone; methylprednisolone; azathioprine;cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine;tizanidine; interferon-β1a (Avonex™; Biogen); interferon-β1b(Betaseron™; Chiron/Berlex): Copolymer 1 (Cop-1; Copaxonem; TevaPharmaceutical Industries, Inc.); hyperbaric oxygen: intravenousimmunoglobulin; clabribine: CDP-571/BAY-10-3356 (humanized anti-TNFαantibody; Celltech/Bayer); cA2 (chimeric anti-TNF antibody; Centocor);75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see e.g.,Arthritis & Rheumatism (1994) Vol. 2, S295; J. Invest. Med. (1996) Vol.44, 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein;Hoffmann-LaRoche); IL-10; IL-4; and IL-10 and/or IL-4 agonists (e.g.,agonist antibodies).

Nonlimiting examples of therapeutic agents for sepsis with which a lowdose of an antibody, antibody portion, or other TNFα inhibitor, of theinvention can be combined include the following: hypertonic salinesolutions; antibiotics; intravenous gamma globulin; continuoushemofiltration; carbapenems (e.g., meropenem); antagonists of cytokinessuch as TNFα, IL-1β, IL-6 and/or IL-8; CDP-571/BAY-10-3356 (humanizedanti-TNFα antibody; Celltech/Bayer); cA2 (chimeric anti-TNFα antibody;Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein;Immunex; see e.g., Arthritis & Rheumatism (1994) Vol. 37, S295; J.Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNFreceptor-IgG fusion protein; Hoffmann-LaRoche); Cytokine RegulatingAgents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); SK&F107647 (low molecular peptide; SmithKline Beecham); tetravalentguanylhydrazone CNI-1493 (Picower Institute); Tissue Factor PathwayInhibitor (TFPI; Chiron); PHP (chemically modified hemoglobin; APEXBioscience); iron chelators and chelates, including diethylenetriaminepentaacetic acid-iron (III) complex (DTPA iron (I); Molichem Medicines);lisofylline (synthetic small molecule methylxanthine; Cell Therapeutics,Inc.); PGG-Glucan (aqeuous soluble β1,3glucan; Alpha-Beta Technology);apolipoprotein A-1 reconstituted with lipids; chiral hydroxamic acids(synthetic antibacterials that inhibit lipid A biosynthesis);anti-endotoxin antibodies; E5531 (synthetic lipid A antagonist; EisaiAmerica, Inc.); rBPI₂₁ (recombinant N-terminal fragment of humanBactericidal/Permeability-Increasing Protein); and SyntheticAnti-Endotoxin Peptides (SAEP; BiosYnth Research Laboratories);

Nonlimiting examples of therapeutic agents for adult respiratorydistress syndrome (ARDS) with which a low dose of an antibody, antibodyportion, or other TNFα inhibitor of the invention can be combinedinclude the following: anti-IL-8 antibodies; surfactant replacementtherapy; CDP-571/BAY-10-3356 (humanized anti-TNFα antibody;Celltech/Bayer); cA2 (chimeric anti-TNFα antibody; Centocor); 75kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see e.g.,Arthritis & Rheumatism (1994) Vol., S295; J. Invest. Med. (1996) Vol.44, 235A); and 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein;Hoffmann-LaRoche).

Other therapeutic agents include chemotherapeutic agents, radiationtherapy, neuroprotective agents and antiinfective agents which may beuseful for treatment of a particular disorder for which TNFα activity isdetrimental.

The language “chemotherapeutic agent” is intended to include chemicalreagents which inhibit the growth of proliferating cells or tissueswherein the growth of such cells or tissues is undesirable or otherwisetreat at least one resulting symptom of such a growth. Chemotherapeuticagents are well known in the art (see e.g., Gilman A. G., et al., ThePharmacological Basis of Therapeutics, 8th Ed., Sec 12:1202-1263(1990)), and are typically used to treat neoplastic diseases. Examplesof chemotherapeutic agents include: bleomycin, docetaxel (Taxotere),doxorubicin, edatrexate, etoposide, finasteride (Proscar), flutamide(Eulexin), gemcitabine (Gemzar), goserelin acetate (Zoladex),granisetron (Kytril), irinotecan (Campto/Camptosar), ondansetron(Zofran), paclitaxel (Taxol), pegaspargase (Oncaspar), pilocarpinehydrochloride (Salagen), porfimer sodium (Photofrin), interleukin-2(Proleukin), rituximab (Rituxan), topotecan (Hycamtin), trastuzumab(Herceptin), tretinoin (Retin-A), Triapine, vincristine, and vinorelbinetartrate (Navelbine).

Other examples of chemotherapeutic agents include alkylating drugs suchas Nitrogen Mustards (e.g., Mechlorethamine (HN₂). Cyclophosphamide,Ifosfamide, Melphalan (L-sarcolysin), Chlorambucil, etc.);ethylenimines, methylmelamines (e.g., Hexamethylmelamine, Thiotepa,etc.); Alkyl Sulfonates (e.g., Busulfan, etc.), Nitrosoureas (e.g.,Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU),Streptozocin (streptozotocin), etc.), triazenes (e.g., Decarbazine(DTIC; dimethyltriazenoimi-dazolecarboxamide)), Alkylators (e.g.,cis-diamminedichloroplatinum II (CDDP)), etc.

Other examples of chemotherapeutic agents include antimetabolites suchas folic acid analogs (e.g., Methotrexate (amethopterin)); pyrimidineanalogs (e.g., fluorouracil (′5-fluorouracil; 5-FU); floxuridine(fluorode-oxyuridine); FUdr; Cytarabine (cyosine arabinoside), etc.);purine analogs (e.g., Mercaptopurine (6-mercaptopurine; 6-MP);Thioguanine (6-thioguanine; TG); and Pentostatin (2′-deoxycoformycin)),etc.

Other examples of chemotherapeutic agents also include vinca alkaloids(e.g., Vinblastin (VLB) and Vincristine); topoisomerase inhibitors(e.g., Etoposide, Teniposide, Camptothecin, Topotecan,9-amino-campotothecin CPT-11, etc.); antibiotics (e.g., Dactinomycin(actinomycin D), adriamycin, daunorubicin, doxorubicin, bleomycin,plicamycin (mithramycin), mitomycin (mitomycin C), Taxol, Taxotere,etc.); enzymes (e.g., L-Asparaginase); and biological response modifiers(e.g., interferon-; interleukin 2, etc.). Other chemotherapeutic agentsinclude cis-diaminedichloroplatinum II (CDDP); Carboplatin;Anthracendione (e.g., Mitoxantrone); Hydroxyurea; Procarbazine(N-methylhydrazine); and adrenocortical suppressants (e.g., Mitotane,aminoglutethimide, etc.).

Other chemotherapeutic agents include adrenocorticosteroids (e.g.,Prednisone); progestins (e.g., Hydroxyprogesterone caproate;Medroxyprogesterone acetate, Megestrol acetate, etc.); estrogens (e.g.,diethylstilbestrol; ethenyl estradiol, etc.); antiestrogens (e.g.Tamoxifen, etc.); androgens (e.g., testosterone propionate,Fluoxymesterone, etc.); antiandrogens (e.g., Flutamide); andgonadotropin-releasing hormone analogs (e.g., Leuprolide).

The language “radiation therapy” includes the application of agenetically and somatically safe level of x-rays, both localized andnon-localized, to a subject to inhibit, reduce, or prevent symptoms orconditions associated with cancer or other undesirable cell growth. Theterm “x-rays” includes clinically acceptable radioactive elements andisotopes thereof, as well as the radioactive emissions therefrom.

Examples of the types of emissions include alpha rays, beta raysincluding hard betas, high energy electrons, and gamma rays. Radiationtherapy is well known in the art (see e.g., Fishbach, F., LaboratoryDiagnostic Tests, 3rd Ed., Ch. 10: 581-644 (1988)), and is typicallyused to treat neoplastic diseases.

Examples of neuroprotective agents include, but are not limited to,compounds that remove protein build up (e.g., geldanamycin),anti-inflammatory agents (e.g., glucocorticoids, non-steroidalanti-inflammatory drugs (e.g., ibuprofen, aspirin, etc.), omega-3 fattyacids (e.g., EPA, DHA, etc.), minocycline, dexanabionol, etc.),compounds that increase energy available to cells (e.g., creatine,creatine phosphate, dichloroacetate, nicotinamide, riboflavin,carnitine, etc.), anti-oxidants (e.g., plant extracts (e.g., gingkobiloba), co-enzyme Q-10, vitamin E (alpha-tocopherol), vitamin C(ascorbic acid), vitamin A (beta-carotene), selenium, lipoic acid,selegine, etc.), anti-glutamate therapies (e.g., remacemide, riluzole,lamotrigine, gabapentin, etc.), GABA-ergic therapies (e.g., baclofen,muscimol, etc.), gene transcription regulators (e.g., glucocorticoids,retinoic acid, etc.), erythropoietin, TNF-α antagonists, cholinesteraseinhibitors, N-methyl-D-aspartate (NMDA) antagonists, opiod antagonists,neuronal membrane stabilizers (e.g., CDP-choline, etc.), calcium andsodium channel blockers, prednisone, etc.

Antiinfective agents include those agents known in the art to treatviral, fungal, parasitic or bacterial infections.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

Example 1 Study of Efficacy of D2E7 Administered in Low Doses

Studies were performed to determine the efficacy of D2E7, infliximab,and etanercept at low doses in preventing polyarthritis using thetransgenic (Tg197) murine model of rheumatoid arthritis (RA). Infliximabis a human-mouse chimeric antibody, and etanercept is a p75 TNF receptorconstruct. D2E7 is a fully human antibody derived from a humanimmunoglobulin gene library. Transgenic mice, Tg197 carry the human TNF□ gene and spontaneously develop a disease similar to human rheumatoidarthritis (Keffer, J. et al, 1991, EMBO J. 10:4025). Signs of arthriticdisease, including rheumatoid arthritis, include slower weight gain,joint distortion and swelling, joint deformation and ankylosis andimpaired movement. Histopathological findings include hyperplasia of thesynovial membrane, leukocyte infiltration, pannus formation, articularcartilage and bone destruction. Administration of anti-TNF agentsprevents the development of polyarthritis in a dose dependent manner.

A. Comparison of Binding Characteristics of D2E7, Remicade, and Enbrel

Infliximab (Remicade) and Etanercept (Enbrel) are two anti-TNF drugsapproved for rheumatoid arthritis. Remicade is a human-mouse chimericIgG₁ antibody and Enbrel is a fusion protein made up of extra-cellulardomain of the p75 TNF receptor and the constant region of IgG₁ molecule.D2E7 is a fully human antibody of the IgG₁,kappa class selected fromhuman immunoglobulin gene libraries. All three anti-TNF agents bind tohuman TNF with relatively similar potency. The intrinsic affinities ofD2E7, Remicade and Enbrel for TNF are 8.6×10⁻¹¹, 9.5×10⁻¹¹ and15.7×10⁻¹¹ M (Kd values), respectively. The kinetics of binding to TNFare similar for the antibodies D2E7 and Remicade. Enbrel, on the otherhand, binds to and dissociates from TNF fast. Thus, the 16 minutehalf-life of Enbrel:TNF complex is considerably shorter than the 184 and255 minute half-lives for Remicade and D2E7:TNF complexes, respectively.

A BIAcore 3000 instrument was used to derive kinetic parameters ofbinding between human TNF and anti-TNF agents. Biosensor chips werecovalently coupled with a goat anti-human Fc antibody. Anti-TNF agents(D2E7, Remicade and Enbrel) were then captured on the chips and varyingconcentrations of huTNF were added. Binding data were analyzed to derivethe kinetic parameters, which are described in Table 1.

TABLE 1 Binding of D2E7, Remicade, or Enbrel to human TNF Agent On-rate(M⁻¹s⁻¹) Off-rate (s⁻¹) Kd (M) D2E7 5.37 × 10⁵ 4.53 × 10⁻⁵ 8.56 × 10⁻¹¹Remicade 6.71 × 10⁵ 6.29 × 10⁻⁵ 9.45 × 10⁻¹¹ Enbrel 4.47 × 10⁶ 7.02 ×10⁻⁴ 1.57 × 10⁻¹⁰

B. Prevention of Arthritic Symptoms

Tg197 Mice were used as a model for studying the effects of a low doseregiment of D2E7, Remicade, and Enbrel on relieving symptoms commonlyassociated with rheumatoid arthritis. Human TNF transgenic mice wereidentified and verified by PCR. From the first week of age, separatelitters of Tg197 mice were assigned to different study groups. Tg197mice heterozygous for the human TNF gene received weekly intraperitonealdoses of D2E7, Remicade or Enbrel. Each drug treatment dose groupconsisted of mice from a single litter. The control group received thephosphate buffered saline diluent and consisted of mice from 4 litters.Weights of animals in each group were recorded weekly prior to dosing.Each group received one i.p. injection per week as follows:

Vehicle Control

D2E7, 10 mg/kg Remicade, 10 mg/kg Enbrel, 10 mg/kg D2E7, 5 mg/kgRemicade, 5 mg/kg Enbrel, 5 mg/kg D2E7, 1 mg/kg Remicade, 1 mg/kgEnbrel, 1 mg/kg D2E7, 0.5 mg/kg Remicade, 0.5 mg/kg Enbrel, 0.5 mg/kgD2E7, 0.1 mg/kg Remicade, 0.1 mg/kg Enbrel, 0.1 mg/kg D2E7, 0.01 mg/kgRemicade, 0.01 mg/kg Enbrel, 0.01 mg/kg

Arthritic scores in each group were recorded each week using thefollowing scoring system:

0 no arthritis 1 mild arthritis (joint distortion) 2 moderate arthritis(swelling, joint deformation) 3 severe arthritis (ankylosis on flexionand severely impairment movement)Treatment continued for 10 weeks. Results from the scoring assay areshown in FIGS. 1, 2, and 3. FIG. 4 shows the final arthritic score forthe three antibodies in treated Tg197 mice at week 10. The results showthat higher doses of Enbrel were needed to prevent the development ofarthritic scores. The ED₅₀ value for Enbrel was close to 1 mg/kg;whereas the ED₅₀ value for D2E7 and Remicade was below 0.5 mg/kg.Between the two antibodies, D2E7 offered more protection than Remicadeat the same doses. Furthermore, the onset of disease was delayed in micetreated with 0.5 mg/kg of D2E7 up to 5 weeks, and with mice treated with0.1 mg/kg doses of D2E7 up to 4 weeks (FIG. 1). In contrast, the onsetof disease was delayed in mice treated with either 0.1 mg/kg or 0.5mg/kg of Remicade for only 3 weeks (FIG. 2).

In sum, all three agents, D2E7, Remicade, and Enbrel prevented thedevelopment of arthritis in Tg197 mice in a dose dependent fashion.Treated mice had lower arthritic scores and less inflammation and jointdamage and gained more weight than the untreated mice. The pattern ofresponse was similar for all three TNF antagonists, but the degree ofprotection varied among the three agents. Although the highest,saturating doses did not allow distinction between the agents, thepotency of protection at intermediate doses was greatest for D2E7treated mice, than for infliximab (Remicade), and the least forEtanercept (Enbrel) treated mice.

C. Analysis of Circulating huTNF levels in Treated Mice

Blood was collected at 5 and 10 weeks during the study. Serum wasprepared and the levels of human TNF were determined by the Medgenixhuman TNF ELISA kit. TNF levels were measured for each mouse intreatment groups. Results from the study are shown in FIG. 6. For eachtreatment group, mean±standard error of TNF levels are indicated in thegraph. A solid line at each graph is drawn at 2 ng/mL TNF level fororientation purpose. In the untreated group, serum huTNF levels werelow, 0.1 and 0.2 ng/mL at 5 and 10 weeks, respectively. Weeklyadministration of anti-TNF agents resulted in sequestration of TNF inthe serum. The levels of serum huTNF were similar for D2E7 or Remicadetreated mice. The average huTNF levels decreased from 2 to 0.1 ng/mL asa function of administered dose. Enbrel treated mice, on the other hand,had much higher serum huTNF, reaching levels of 20 ng/mL.

In sum, measurement of human TNF by Medgenix ELISA, which detects bothfree and bound TNF, indicated that the anti-TNF agents were sequesteringTNF into complexes. There were detectable levels of TNF in the serum oftreated mice in contrast to very low levels in untreated mice.Interestingly, the level of noncleared TNF complexes for etanercept was10-fold higher than in mice treated with infliximab or D2E7. Delayed TNFclearance with etanercept has been noted in published animal models andclinical studies.

D. Microscopic Analysis of Treated Mice

Following the 10 week treatment, all mice were sacrificed. Right andleft hind limbs were harvested from two mice in each treatment group.Limbs were fixed in 10% neutral buffered formalin and then decalcified.Three consecutive sections from each limb sample were mounted on slidesand the coded slides were sent for an independent evaluation by apathologist.

Slides were stained with hematoxylin/eosin. The pathologist scored eachslide with respect to severity of vascularity, inflammation, cartilageand bone erosion on a scale of 1-4. Results are shown in Table 2 below:

TABLE 2 Approximate ED₅₀ (mg/kg) values of D2E7, Remicade or Enbrel forprevention of microscopic signs of arthritis in Tg197 mice D2E7 RemicadeEnbrel ED₅₀, mg/kg ED₅₀, mg/kg ED₅₀, mg/kg Inflammation 0.1 0.5 0.5Vascularity 0.1 0.1   1 < ED₅₀ < 5 Cartilage 0.01 < ED₅₀ < 0.1 0.1 <ED₅₀ < 0.5 0.5 Erosion Bone Erosion 0.01 < ED₅₀ < 0.1 0.1 < ED₅₀ < 0.50.5 < ED₅₀ < 1

Results from this experiment are also shown in FIG. 5. In FIG. 5, threeslides from each limb were examined; thus, 6 slides per mouse and 12slides per treatment group were scored for histopathology. For eachtreatment group, mean t standard deviation of histopathology scores areindicated in the graph. Most of the lesions were associated with anklejoints and all appeared symmetrical (i.e. similar scores for the leftand right limbs for a given mouse). Cartilage degradation resultedmostly from endosteal erosive lesions and was generally less extensivethan bone erosion. Inflammation was predominantly mononuclear cells withfew PMNs, but no dense PMN loci.

The difference among the three anti-TNF agents was most pronounced inmicroscopic signs of disease activity in the arthritic joints than theexternal manifestations measured as arthritic scores. Bone erosion inthe joints was completely abolished by 0.5 mg/kg dose of D2E7. In orderto achieve the same effect a much higher dose of Remicade or Enbrel, 5mg/kg, was needed. Cartilage erosion in the joints was completelyabolished by 0.1 mg/kg dose of D2E7. In order to achieve the same effecta much higher dose of Remicade, 1 mg/kg, or Enbrel, 5 mg/kg, was needed.Inflammation in the joints was completely abolished by 0.5 mg/kg dose ofD2E7. In order to achieve the same effect higher doses of other drugswere needed: 5 mg/kg for Remicade and 10 mg/kg for Enbrel. Vascularityin the joints was completely abolished by 0.5 mg/kg dose of D2E7 orRemicade. In order to achieve the same effect a much higher dose ofEnbrel, 5 mg/kg, was needed.

There was a clear dose-response distinction between D2E7, infliximab andetanercept in prevention of microscopic joint damage. Whereas D2E7completely prevented bone erosion, cartilage degradation, inflammation,and vascularity at the 0.5-mg/kg dose, both infliximab and etanerceptrequired a dose of 1 or 5 mg/kg to reach similar levels of efficacy. Insum, in the human TNF transgenic mice, Tg197, D2E7 preventedpolyarthritis more potently than did etanercept or infliximab,especially at low doses.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. The contents of allreferences, patents and patent applications cited throughout thisapplication are hereby incorporated by reference. The entire contents ofU.S. Pat. Nos. 6,090,382 and 6,258,562 B1, and in U.S. patentapplication Ser. Nos. 09/540,018, and 09/801,185, are herebyincorporated herein by reference in their entirety.

1. A human anti-TNFα antibody which is either D2E7, or a human anti-TNFαantibody with equivalent properties to D2E7 which dissociates from humanTNFα with a K_(d) of 1×10⁻⁸ M or less and a keg rate constant of 1×10⁻³s⁻¹ or less, both determined by surface plasmon resonance, andneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻⁷ M or less, for use in the treatment oralleviation of arthritis, wherein the anti-TNFα antibody is to beadministered in a low dose of 0.01-0.11 mg/kg, such that the arthritisis treated or alleviated.
 2. The human anti-TNFα antibody according toclaim 1, for use in the treatment or alleviation of rheumatoidarthritis.
 3. The human anti-TNFα antibody according to claim 1, whereinsymptoms associated with the arthritis are alleviated.
 4. The humananti-TNFα antibody according to claim 1, wherein symptoms selected fromthe group consisting of bone erosion, cartilage erosion, inflammation,and vascularity are alleviated.
 5. The human anti-TNFα antibodyaccording to claim 1, wherein the arthritis is treated by alleviatingsymptoms selected from the group consisting of joint distortion,swelling, joint deformation, ankylosis on felxion, and severely impairedmovement.
 6. The human anti-TNFα antibody according to claim 1, whereinthe antibody sequesters TNFα into complexes in a subject suffering fromsaid arthritis.
 7. The human anti-TNFα antibody according to claim 6,wherein the serum level of TNFα is higher than the serum level of TNFα asubject not suffering from said arthritis.
 8. The human anti-TNFαantibody according to claim 1, which is D2E7.